Program Director/Principal Investigator (Last, First, Middle): Ren, Dejian The overall goal of the proposed research is to understand the function and the regulation of potassium ion channels to the function of lysosomes. Potassium is the most abundant intracellular ion that faces both plasma membrane and organelle membrane. Plasma membranes are highly permeable to potassium. More than 80 potassium channels have now been discovered to mediate such plasma membrane potassium permeability. In contrast, how potassium passes intracellular organelles such as lysosomes is much less understood. Lysosomes play fundamental roles in cellular clearance, digestion, recycling, exocytosis and membrane repair. Because of high concentration of calcium in the lumen, lysosomes are also calcium stores from which calcium is released into cytosol to shape cytosolic calcium kinetics and to regulate muscle contraction and hormone secretion. Lysosomal dysfunction has been linked to pathophysiological conditions such as lysosomal storage diseases, cancer and neurodegeneration. Therefore understanding how lysosomal membrane mediates ionic permeability is important to our understanding of the organelle?s physiology function. We recently discovered a novel protein TMEM175 that forms a potassium-selective channel in lysosomal membrane. Genetic variation in human TMEM175 is also implicated in Parkinson?s disease. We propose three specific aims to expand our preliminary findings. In Aim 1, we will use patch clamp recordings to compare wild-type and TMEM175 knockout animals and will test whether there are other major lysosomal potassium channels in addition to TMEM175. Unlike those of plasma membrane channels, the functions of lysosomal potassium channels are largely unknown. In Aim 2, we will use the TMEM175 knockout mouse model to test the hypothesis that potassium channels regulate the normal working of lysosomes including organelle membrane potential regulation, luminal acidification, vesicle fusion and autophagy. In Aim 3, we will test whether TMEM175, like many other potassium channels on plasma membrane, is regulated by protein kinases. Using protein chemistry experiments, we will also determine the structural requirements for such regulation. Because of the fundamental function of lysosomes, the studies will reveal how lysosomal potassium channels contribute to the organelle?s function under physiological and pathological conditions. OMB No. 0925-0001/0002 (Rev. 01/18 Approved Through 03/31/2020) Page